Stable isotopes reveal seasonal dietary responses to agroforestry in a venomous mammal, the Hispaniolan solenodon (Solenodon paradoxus)

Abstract While trends in tropical deforestation are alarming, conservation biologists are increasingly recognizing the potential for species survival in human‐modified landscapes. Identifying the factors underlying such persistence, however, requires basic ecological knowledge of a species’ resource use. Here, we generate such data to guide conservation of an understudied venomous mammal, the Hispaniolan solenodon (Solenodon paradoxus), that occupies a mosaic landscape of agriculture and forest fragments in the western Dominican Republic. Using feces collected in both wet and dry seasons, we found significant differences in the stable isotope values of carbon (δ13C) between pasture (−24.63 ± 2.31‰, Las Mercedes) and agroforestry (−28.07 ± 2.10‰, Mencia). Solenodon populations in agricultural areas occupied wider isotopic niche spaces, which may be explained by more diverse resource within these patches or individuals combining resources across habitats. We detected elevated δ15N values in the dry season of pasture areas (8.22 ± 2.30‰) as compared to the wet season (5.26 ± 2.44‰) and overall narrower isotopic niche widths in the dry season, suggestive of the impacts of aridity on foraging behavior. Our work highlights the importance of considering a more nuanced view of variations in ‘modified’ or “agricultural” landscapes as compared with strictly protected national parks. We suggest that seasonal differences in foraging should be considered as they intersect with landscape modification by landowners for maintaining resources for focal consumers. This work adds to a growing body of literature highlighting that fecal stable isotopes are a non‐invasive and cost‐effective monitoring tool that is particularly well‐suited for cryptic small mammal species, ensuring actionable and evidenced‐based conservation practices in the tropic's rapidly changing landscapes.


| INTRODUC TI ON
The tropical landscapes of the future will look different from those of the recent past, consisting of vegetation mosaics that reflect varying levels of anthropogenic activity and economic pressures (Mendenhall et al., 2014;Pendrill et al., 2019;Tilman et al., 2017). A growing body of research suggests that certain species may survive and even thrive in these human-dominated landscapes based on their traits and evolutionary history (Frank et al., 2017;Hirschfeld & Rödel, 2017). Many of these attributes-trophic position, dietary flexibility, feeding guild, niche breadth-are intimately linked to how species use resources (Solari et al., 2016). Assessing whether a species' resource base is impacted by land use change, and if so, whether the species can utilize these new resources, can provide critical planning insight for supporting species and populations that occur outside of core protected areas in the tropics .
While in general small mammals are expected to be less vulnerable to extinction than larger mammals (Cardillo et al., 2008), many species have restricted geographic ranges that can be easily wiped out by landscape alterations (e.g., Cervantes & Guevara, 2010) and island endemics have unique evolutionary histories with additional vulnerabilities, such as reduced reproductive rates (Lyons et al., 2016). The present-day "security" of small mammal conservation risk contrasts sharply with the massive Holocene extinctions of insular small mammals-many associated with colonial agricultural practices (Cooke et al., 2017). For example, the Caribbean was once a hotspot of small mammal evolutionary diversity, but of the ~130 described species just 13 terrestrial mammals and 60 bats persist today following multiple waves of anthropogenic extinction both before and after European arrival (Turvey & Fritz, 2011;Turvey et al., 2017).
Here, we evaluate how landscape change on the Caribbean island of Hispaniola (Dominican Republic and Haiti) is impacting one of the island's two remaining endemic terrestrial mammals, the Hispaniolan Solenodon (Eulipotyphla: Solenodon paradoxus).
The solenodon is a nocturnal species so elusive that it was once considered easier to find a "ghost" (Verrill, 1907), and the lack of natural history data hinders assessment of their conservation needs (Rupp & Leon, 2009). If phylogenetically conserved, the diet of solenodons should resemble its relatives such as hedgehogs, which rely heavily on abundant invertebrate prey, supplemented occasionally by vertebrate prey and plants . On the other hand, the legacy of millions of years of insular evolution has resulted in a departure of life history traits from the typical small-bodied mammalian insectivore that breeds rapidly, has large litter sizes, and has a short lifespan (Symonds, 1999): the solenodon attains a relatively large body size of ~1 kg, births only two-yearly litters of one to two young with a prolonged lactation period of 2-3 months, and is social, with family groups of up to six individuals sharing a burrow (Casewell et al., 2019;Eisenberg, 1975;Ottenwalder, 1991). Thus, the solenodon potentially has characteristics of both vulnerable species (slow reproductive rate, restricted distribution, evolution in absence of mammalian carnivores) and resilient species (a generalist, omnivorous diet, and nocturnal habits) (Liow et al., 2009). Virtually all known descriptions of the species' feeding ecology are derived from captive conditions in which they have typically been fed a diet heavy in vertebrate-derived proteins such as horse meat, beef, 2-to 3-day-old chicks, skinned mice, eggs, and milk (Ottenwalder, 1991(Ottenwalder, , 1999; Allen (1910) notes that "they eat greedily of chopped meat." However, the majority of these items would not naturally be encountered, and the small number of wild solenodon observations instead emphasize a diet of invertebrates, such as Verrill's (1907) early description of the animal as "rooting in the earth and cultivated grounds, tearing rotten logs and trees to pieces with its powerful front claws, and feeding on ants, grubs, insects, vegetables, reptiles, and fruit… on several occasions it has been known to enter the houses in search of roaches and other vermin." Accounts of the solenodon's extant sister lineage, the Cuban almiqui (Atopogale cubana), similarly emphasize a reliance on insects and worms in the wild (Echenique-Díaz et al., 2014).

Recent habitat surveys have observed solenodons in forest
fragments both within and outside of protected areas throughout the island of Hispaniola . Sixty percent of the Dominican Republic's original forest was cut between 1930 and 1980 CE and replaced by oil palm, sugarcane, cacao, pastures, coffee, and human settlements (Bolay, 1997). Recent political instability and natural disasters have led to significant increases in human movement from Haiti to the Dominican Republic, resulting in uneven deforestation and disturbance both near and within national parks as a result of illegal charcoal harvest, fire, and small-scale subsistence agriculture (Lloyd & León, 2019;Pasachnik et al., 2016). These hotspots of rapid and unregulated deforestation overlap with one of the last remaining refuges for the solenodon, the La Selle-Bahoruco-Jaragua-Enriquillo UNESCO Biosphere Reserve .
Stable isotopes provide an important toolkit to elucidate how species diets and interactions respond to deforestation and disturbance. Carbon stable isotopes (δ 13 C) reveal differences in how species rely on C3 plants (Calvin-Benson cycle, δ 13 C value range −35 to −20‰) or CAM/C4 plants (Crassulacean acid metabolism/Hatch-Slack pathway, δ 13 C value range −15 to −7‰), and thus whether they move between open and closed habitats (Ben-David & Flaherty, 2012;DeNiro & Epstein, 1978;Giroux et al., 2015). Nitrogen is enriched with each trophic transfer upwards to consumers (Vanderklift & Ponsard, 2003) and thus stable nitrogen isotopes (δ 15 N) can illustrate the trophic position of an organism within its food chain, but also can reflect its level of nutritional stress (McCue & Pollock, 2008) and aridity conditions (Styring et al., 2016). In forested environments (e.g., preserves and fragments) we would expect to identify consumption of C3 plants (more negative δ 13 C values) and an intact, complex trophic structure that has stepwise increases in δ 15 N from herbivores to top predators following typical bioaccumulation processes. In areas that are modified and/or have agricultural activities, studies have detected incorporation of C4 foods as either crops or agricultural weeds (more positive δ 13 C values) and a modified trophic structure in which δ 15 N values do not increase in a stepwise fashion, but instead fluctuate depending on the anthropogenic food that consumers opportunistically eat and whether it has been supplemented by fertilizers (White et al., 2012). Combining foods from multiple vegetation and potential fertilizer baselines in humanmodified landscapes can result in consumer populations that have more individually variable diets, and as a result, occupy a wider isotopic niche space than populations of the same species occupying intact landscapes (Magioli et al., 2019).
We used fecal stable isotopes (δ 13 C and δ 15 N) to characterize solenodon foraging behavior across seasons and land use types in a forest-agricultural mosaic landscape (Figure 1a). We assessed dietary variation both across seasons and across forest, pasture, and cropland sites, and explored whether agricultural populations exhibited greater isotopic niche widths as a reflection of increased resource heterogeneity with access to different invertebrate prey.
We opportunistically processed historical solenodon hairs as a reference set of the natural range of variation for a more typically used tissue and to provide a foundation for future isotopic studies as a comparative baseline of anthropogenic conditions. Together, these data represent a window into solenodon diet and behavior of immediate application to both in and ex situ conservation efforts, as well as a monitoring strategy that can be applied to other elusive species globally.  (Ottenwalder, 1999). Villages in the area rely on subsistence agriculture, animal husbandry, and small-scale cash F I G U R E 1 (a) An extremely fresh solenodon fecal sample. Typical signs of solenodon foraging include digging in leaf litter (b) and rotten logs (c) with their probing, ball-and-socket jointed nose and robust and clawed forelimbs. (d) A Cuban almiqui (Atopogale cubanus) at the Smithsonian National Museum of Natural History-this species is extremely rare in museum collections Endemic plants clustered on exposed limestone and variable canopy height with epiphytes and lianas (Fisher-Meerow & Judd, 1989;León et al., 2011;Ottenwalder, 1999  Though we group localities within our focal region into "agriculture"

| Sampling area and local vegetation
and "forest" for ease of contrast, these are relative terms, as the entire area is considered disturbed and used by humans outside of the strict core protected area of the UNESCO Biosphere Reserve; therefore, our analyses compare degrees of disturbance within an overall altered landscape.

| Feces collection and processing
Solenodons can be found living in groups within limestone karst burrows and holes within dead trees, with multiple related individuals using the same landscape in an average home range of ~156,700 m 2  Table S1 for average environmental conditions).
We identified active solenodon foraging sites using "nosepokes," which are conical holes in the soil and leaf litter (Figure 1b), as well as other foraging modes such as rooting through rotting wood ( Figure 1c). The social behavior of solenodons in foraging as a group, sharing a burrow, and defecating together suggests that we captured feces from multiple individuals (Kennerley, 2014;Ottenwalder, 1991). Our fecal sampling approach was designed to minimize the potential for pseudoreplication by collecting fresh feces from a single area within the same day, rather than returning to the same burrow over multiple days, such that a single individual would not be producing the same fecal samples over time. Each fecal sample was categorized as being from an "agriculture" or "forest" context upon collection based on local conditions-for example, the nearby presence of livestock, crops, or recent preparation of soil for planting through burning or tilling.
Fifty fecal samples were stored with silica beads and the outside layer was removed during processing to avoid surface contaminants.
Feces were then ground to a fine powder and homogenized using a mortar and pestle. We analyzed fecal isotopes at the population level as grouped by landscape category and season, rather than focally following individuals, consistent with prior isotopic monitoring of wild mammals (Blumenthal et al., 2012;Phillips & O'Connell, 2016).

| Historical hair samples
We sampled guard hairs from 32 individuals from several mu- We washed ~1 mg of hair in a 2:1 chloroform/methanol solution to remove lipids and contaminants, followed by methanol and water (Oelze, 2016). After decanting, hairs were dried under a fume hood for 48 h.

| Stable isotope analysis
Samples were packed into 3.5 × 5 mm tin capsules and flash combusted on a Thermo Finnigan Deltaplus XL interfaced with a Costech Environmental Analyzer at Stanford University's Stable Isotope Biogeochemistry Lab. We used multiple standards within each run, including both in-house laboratory standards of acetanilide and bovine gelatin powder, and the international standard USGS 40 (l-glutamic acid). We determined an external precision of <0.1‰ for δ 15 N and <0.15‰ for δ 13 C and internal precision of <0.3‰ for δ 15 N and δ 13 C. We included at least four blanks per run to detect contamination.
All results are reported using the δ notation with values in parts per mil (‰) relative to an international carbon (VPDB) and nitro- Anthropogenic fossil fuel use has depleted the δ 13 C values of atmospheric CO 2 relative to pre-Industrial levels; therefore, we corrected all samples to 2015 with a year-specific depletion value (Long et al., 2005) to account for the Suess effect when comparing samples collected at different times. Raw stable isotope values are reported in Table S2.

| Statistical analyses
We used standard tests of normality, including Shapiro-Wilk tests to assess normality and F tests to assess variances for our δ 13 C and We calculated niche width for different groups within sites (dry vs. wet season, forest vs. agriculture) using two metrics: the total convex hull area (TA) and the standard ellipse area (SEA) with the R package Stable Isotope Bayesian Ellipses in R, 'SIBER' (Jackson et al., 2011). TA measures niche width by drawing a convex hull containing all points within a group plotted in a δ 13 Cδ 15 N biplot and is sensitive to differences in sample size (Layman et al., 2007).
SEA (small sample size corrected, SEA c ) is not sensitive to sample size, and calculates the standard ellipse that contains ~40% of the data (comparable to standard deviation in univariate calculations), measured in per mil squared (‰ 2 ) (Jackson et al., 2011). We also calculated ellipses scaled to represent a 95% confidence ellipse of the bivariate means.
We then fit Bayesian multivariate normal distributions to each group and its posterior distribution, facilitating comparison of SEA c area between groups (n = 10,000, burnin = 1000). To test whether ellipses (and therefore isotopic niche widths) were different between groups, we calculated the probability that the posterior distribution of a group's SEA c is larger or smaller than the other: we compared the posterior draws for both groups and calculated the proportion of draws that were smaller as a proxy for the probability that one group's posterior distribution is smaller than the other (based on 10,000 draws).
In addition to calculating TA and SEA c for groups, we determined δ 13 C Range (CR), δ 15 N Range (NR), Mean Distance to Centroid (CD), and Nearest Neighbor Distance (NND) to assess variation (Layman et al., 2007). We used SIBER to calculate these community ecology metrics, which implements a Bayesian inference technique to incorporate the uncertainty of centroid location in our focal communities.
As defined in Layman et al. (2007), CR is calculated as the difference between the highest and lowest values of δ 13 C and reflects the diversity of primary producer resources, though caution should be taken in considering fractionation effects between trophic positions.
NR is calculated as the difference between the highest and lowest values of δ 15 N and reflects the trophic diversity or trophic length in a community. CD is used to evaluate spacing within the niche. NND is a measure of clustering within a community, and SDNND can be similarly informative regarding variability of individual resource use.

| Environmental, anthropogenic, and climatic variables
Using R packages "sp," "raster," "maptools," and "dismo," we extracted values from several datasets for the specific coordinates of each fecal sample, including: percent forest cover (satellite images of tree canopy, 30 m 2 resolution (Hansen et al., 2013)), human population density (National Statistics Office of the Dominican Republic, township/municipality level, 1 km 2 resolution), soil (SoilGrids, 4.5 km 2 resolution (Leenaars et al., 2017)), geology (1:25,000,000 scale; USGS (French & Schenk, 2004)), topography (elevation, slope, aspect; USGS, ESRI), and land use type (Global Land Cover 2000, 1 km 2 resolution (Fritz et al., 2003)). We compared these layers with our on-the-ground categorization of presence of cultivated crops/ pasture and recent Google Earth Satellite Imagery (2017). We selected bioclimatic variables (~1 km 2 resolution) of known importance to solenodon habitat selection based on a prior species distribution modeling study in the region (Gibson et al., 2019), including mean diurnal range (Bio2), temperature seasonality (Bio4), max temperature of warmest month (Bio5), temperature annual range (Bio7), precipitation seasonality (Bio15), and precipitation of warmest quarter (Bio18) (Hijmans et al., 2005). To compare wet and dry season samples, we also extracted sample-specific values of WorldClim's minimum temperature (T min ), maximum temperature (T max ), average temperature (T avg ), and precipitation for the collection month. We used a MANOVA to compare these environmental, anthropogenic, and climatic variables between sampling site locations (Mencia vs. Las Mercedes) and land use categorizations (agriculture vs. forest).
We incorporated temperature and precipitation values at time of sampling, rather than categorizing samples as "dry" or "wet" season.
To explore factors that may influence solenodon isotopic values, we conducted stepwise regressions in R for δ 15 N and δ 13 C using the R package "MASS" using both a forward and backward search, which at each step calculates an AIC value and either includes or excludes a variable, until the best fit and most parsimonious model is found.
We then used the R package "stargazer" (Hlavac, 2018) to summarize the resulting models.

| Sampling site characteristics
Las

| Comparing hair and fecal isotopes
Hairs ranged from δ 13 C −20.58 to −24.55‰ and δ 15 N 5.65 to 9.11‰ from localities across the Dominican Republic, with a low δ 15 N value from Haiti of 3.79‰ (Table 1)

| Seasonal patterns in Las Mercedes
We collected 14 dry season (average δ 15 N 8.09 ± 1.74‰ and δ 13 C −25.35 ± 2.59‰) and 24 wet season (average δ 15 N 6.81 ± 2.86‰ and δ 13 C −26.23 ± 2.49‰) fecal samples within the area of Las Mercedes, across the properties of several landowners (Figure 3a,b; Table 2). Both TA and SEA c were consistently larger for agricultural sites as compared with forest sites across seasons (Figure 3c; Table 3). The agricultural sites across seasons also exhibited a larger δ 13 C and δ 15 N range, CD, NND, and SDNND, suggesting more variation in individual resource use (Table 3). We found that the SEA b (Bayesian SEA estimate) is larger for the wet season both for agriculture (.7868 probability) and forest (.6275 probability) (Figure 3d).
We found significant differences in δ 15 N values across the wet and dry seasons of agriculture and forest fecal samples in Las Mercedes

| Correlates of isotopic values
We used Pearson correlation of r 2 < .70 (Merow et al., 2013;Warren et al., 2010) to remove correlated variables and avoid collinearity ( Figure S2). This left slope, aspect, Bio18, Bio2, Bio15, Bio4, mean seasonal temperature, forest cover, and human population density for use in a stepwise regression. Stepwise regressions of the best models returned by the selection procedure for δ 13 C (AIC = 90.54) and δ 15 N (AIC = 81.9) exhibited low r 2 values (.195 and .288, respectively), though identified several significant coefficients (Table S3).

| DISCUSS ION
The ability of species to persist in new ecological configurations will depend on their individualistic responses to changes in resource availability, including dietary flexibility and foraging behaviors (Castaño-Villa et al., 2019), as well as tolerance to disturbance and human-wildlife conflicts . Understanding the determinants of persistence in turn can inform monitoring strategies and help identify conservation priorities outside of protected areas and in agroforestry landscapes, where significant biodiversity conservation opportunities exist (Cassano et al., 2014;Martin et al., 2020).
Without basic natural history knowledge, we cannot adequately anticipate species responses to land use change. Based on evolutionary history, we hypothesized that the solenodon would be a generalist insectivore across varying landscapes; however, its relatively large body size (~1 kg) and potential consumption of poultry may make it more prone to targeted killings by farmers, thus changing the calculus of foraging risk for solenodons in human-altered spaces.
Historic hair samples from museum collections were consistent with published isotope ranges for insectivorous American shrew species (Baltensperger et al., 2015) and South American shrew mice and opossums (Galetti et al., 2016). In the absence of information on contemporaneous land use conditions, we are unable to meaningfully interpret the ecology of historical solenodon populations based on their hair alone, but we present these values to (1) assess general congruence with fecal isotope values and (2) provide references for future conservation work with these populations in a TA B L E 2 Summary of stable isotope sample sizes (n), values of nitrogen (δ 15 N), carbon (δ 13 C), and standard deviations (SD), for solenodon fecal samples analyzed in this study Abbreviations: CD, mean distance to centroid; CR, carbon range; NND, nearest neighbor distance; NR, nitrogen range; SDNND, standard deviation of nearest neighbor distance; SEAc, standard ellipse area small sample size corrected; TA, total area. We used feces to provide isotopic snapshots of resource use and may alter their responses to disturbance and landscape modification on highly local and seasonal scales dependent on types of agricultural activity. Fecal stable isotopes have been shown to reliably distinguish between pure C3, pure C4, and mixed feeder diets (Codron et al., 2005). Our estimates of the δ 15 N and δ 13 C resources used by solenodon populations sampled via hair and feces (corrected for trophic discrimination) (Montanari, 2017;Salvarina et al., 2013;Siemers et al., 2011) broadly range from ~3 to 6‰ and ~−26 to −30‰, respectively. These values are consistent with a mostly C3 primary productivity baseline as expected for the type of anthropogenic landscape change in our region of study. While humans have been altering Hispaniolan landscapes since the mid-Holocene (Cooke et al., 2017), the majority of stable isotope research for the region has focused on archaeological and paleontological-rather Studies of a diverse range of taxa, including Malaysian rodents (Nakagawa et al., 2007), lemurs (Crowley et al., 2013), pumas (Magioli et al., 2014), Borneo bird communities (Hamer et al., 2015), bats (Reuter et al., 2016), and even ant colonies (Woodcock et al., 2013) have reported increased δ 15 N values associated with disturbed and/ or degraded forest ecosystems. We did not detect significant differ-  -Meerow & Judd, 1989;León et al., 2011;Ottenwalder, 1999 applying the label to any location outside of a strictly protected area, as these patches may be managed differently by different landowner preferences and local topographical conditions, facilitating different population outcomes even within "buffer" areas outside of national parks (Daily et al., 2003;Marín et al., 2009).

Our analysis of seasonal change across wet and dry seasons in Las
Mercedes highlighted how landscape modification may interact with seasonal conditions to shape foraging behaviors (e.g., Teodoro et al., 2010 we found that the isotopic niche of the wet season was broader than the dry season. Evidence for larger wet season home ranges in solenodons is consistent with previous observations that solenodon above-ground activity decreases during the dry season, possibly due to reduced abundance or diversity of invertebrate prey, conservation of energy, and a peak in breeding efforts (Ottenwalder, 1991  . While these matrices may harbor lower species richness (Bogoni et al., 2017), they act as feeding areas for a number of species (Magioli et al., 2019). It is important to caution that while such modified areas may provide resources for solenodons and other species more broadly in the tropics, these are also spaces with greater numbers of human commensal species such as dogs, and higher levels of human-wildlife conflict for certain taxa, especially if they are considered threats to livestock or crop production, potentially negating the benefits of resource availability (Crespin & Simonetti, 2019).
The pace of deforestation in tropical landscapes requires methodological toolkits of varying temporal scales to capture both initial and long-term alterations in foraging ecology and habitat use. Fecal isotopes can provide a nearly instantaneous understanding of what resources an organism is utilizing as well as the variation between individuals foraging at the same time within and between landscapes (Blumenthal et al., 2012;Phillips & O'Connell, 2016). This timescale may be especially appropriate in rapidly shifting and regenerating mosaic landscapes, such as the areas where some of the last solenodon populations persist despite burning of forests for charcoal and sharecropping (Lloyd & León, 2019). Fecal isotopes can be an important component of the practitioner's toolbox for monitoring populations and detecting potential threats or declines as disturbances unfold. Fully realizing the potential of this tool in broader conservation contexts requires future controlled feeding studies in captivity to determine Δ diet-feces for a wider range of taxa (e.g., as in Montanari, 2017), as these trophic enrichment factors are necessary for isotopic mixing models and delimiting species interactions. Fecal isotope analysis can be expanded as a viable monitoring strategy for solenodons moving forward across Hispaniola's diverse environments and land use systems. We recommend such an approach for the Caribbean's other last surviving mammals, such as the Hispaniolan hutia, which are in urgent need of basic natural history studies and enhanced monitoring efforts before they are no longer considered "Last Survivors" but, rather, recent casualties.

ACK N OWLED G M ENTS
This work would not have been possible without the contributions of local field assistants (Yimell Corona, Nicolas Corona, Jose Ramon "Moncho" Espinal, Gerson Feliz) and landowners (especially. Sr. Cigua). All samples were collected non-invasively with permission from the Departamento de Investigaciones de la Subsecretaria

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest associated with this study. (2018). Stable isotope analysis of fecal material provides insight